The present invention is directed to an injectable, biocompatible and biodegradable composition, comprising at least one hyaluronic acid benzyl ester or auto-cross-linked derivative, in combination with at least one mammalian cell and/or at least one pharmacologically or biologically active substance and/or micro-particles such as fibres, granules, microspheres or sponge fragments of a hyaluronic acid derivative.
Although injectable compositions and carriers for such compositions have been known in the art, there still exists a need for injectable compositions which are biocompatible, are biodegradable, offer protective aspects to the active component, and provide enhanced bioavailability of the active components. This is important, for instance, in the field of joint cartilage repair.
The aim of joint cartilage repair is to restore the surface of the joint, reduce pain and prevent further deterioration of the tissues. Many methods have been applied to date for the treatment of cartilage defects, each of which has presented disadvantages (Tom Minas et al., xe2x80x9cCurrent Concepts in the treatment of Articular Cartilage Defectsxe2x80x9d, Orthopedics, June 1997, vol. 20, No. 6).
The marrow stimulation technique consists of reaching subchondral bone tissue areas by means of abrasion or perforation, thus stimulating the formation of a fibrin 30 clot containing pluripotent stem cells. The clot subsequently differentiates and takes shape, forming fibrocartilage repair tissue. However, this tissue does not have the mechanical properties or the physiological and structural characteristics of healthy, lasting joint cartilage.
Another technique consists of implanting into the site of the defect a piece of periosteal and perichondral tissue taken, for example, from the rib cartilage. Such treatment does trigger the development of hyaline cartilage, but the repair tissue is poorly integrated with the surrounding healthy tissues and the implanted tissue subsequently becomes ossified.
Autologous and homologous osteochondral grafts are invasive, require complex surgical techniques and carry the risk of, for example, viral infection.
Other attempts to reconstruct the joint cartilage consist of implanting synthetic matrices with allogenic chondrocytes dispersed within them, or growth factors able to stimulate the proliferation of the chondrocytes. These methods require that the cartilage tissue is grown in vitro and then implanted into the defect. The synthetic matrices most commonly used are collagen gels, matrices of polyanhydrides, polyorthoesters, polyglycolic acid and its copolymers. The chief disadvantage of the use of such matrices is represented by the immune response directed against the implanted material. Chondrocytes are known to be cultured in gel constituted by agarose, hyaluronic acid, fibrin glue, collagen and alginate. However, these cultures in gel do not provide the mechanical stability necessary for them to adhere to the site once implanted and to allow the reconstruction of the cartilage structure. Moreover, chondrocyte cultures in substances such as fibrin de-differentiate into cells which appear to be similar to fibroblasts. Lastly, although gels constituted by substances such as agarose induce chondrocyte re-differentiation, the use of this compound has not been approved for internal applications to humans.
Joint cartilage defects have also been treated with suspensions of isolated chondrocytes in the absence of supporting matrices. It is thought, however, that chondrocytes lose their viability and/or do not remain at the site of the defect and that they form fibrocartilage or islets of cartilage immersed in fibrous tissue (see U.S. Pat. No. 5,723,331).
Some biological materials consisting of hyaluronic acid derivatives have been used to fabricate porous degradable scaffolds for tissue repair, reconstruction and wound healing (WO 97/45532). Others have been shown to support the growth of poor resistant and weak cells (WO 98/56897). These materials, however, are not injectable.
These disadvantages of the prior art are overcome by the present invention by providing an injectable composition such as one containing chondrocytes or bone marrow stroma cells dispersed in a gel containing at least one hyaluronic acid benzyl ester derivative or auto-cross-linked derivative.
Various pieces of evidence have emerged in the literature (see enclosed abstract) recently concerning the use of cell suspensions for injection purposes, in particular keratinocytes for the treatment of chronic ulcers and burns. See Silverman et al, Plast. Reconstr. Surg., June 1999, 103(7) 1809-18 (combination of fibrinogen and chondrocytes); Atala et al., J. Urol., Aug. 1993, 150 (2 Ptd. 2) p. 745-7 (chondrocyte-alginate gel)). Keratinocyte cultures can be developed according to various methods cited in the literature (in the presence or absence of foetal calf serum, with chemically defined culture medium, etc.). These cultures are then vehicled in the host bed suspending them in various media, one of the most frequently cited of which is fibrin both of autologous and commercial origin. There are considerable disadvantages to the use of such methods. Firstly, the cell suspension has to be prepared immediately before use, so the cells have to be stored in a medium with a different composition from the one used for their application, while other problems may arise with the fibrin glue used as a vehicle, particularly when this is not autologous.
These problems are overcome by the present invention by dispersing epithelial cells (such as keratinocytes) or derivatives of other embryonic origin in a hyaluronic-acid-based medium for various reasons. The preparation is perfectly biocompatible and biologically safe and the cell survival rate is higher than in cell suspensions in completely liquid media. This last point in particular is important. In cases where the patient or application site is a long distance from the site of production for the component, safe transport becomes a problem. The product will inevitably be shaken about during transport damaging the cells, and this problem needs to be solved. However, when the cells are dispersed in a highly viscous medium according to the present invention, this problem is overcome because the host medium acts as a cushion. Another advantage derives from the possibility of spreading the cell suspension efficiently over the surface to be treated, which is a simpler way of applying it than the methods currently used, involving sprays based on fibrin glue.
Another application of the present invention concerns the possibility of suspending the cells in the medium and then applying them by injection. Other non-limiting applications are the administration of fibroblasts (autologous) for aesthetic surgical purposes or as fillers for tissue defects, preparations of adipocytes (autologous, heterologous or homologous) for soft tissue augmentation for applications such as the reconstruction of breasts or other soft body parts, injections of urethral cells such as fibroblastoids or cartilage cells for the treatment of urinary incontinence. In all these examples, the Hyaluronic acid-based material has the double function of acting as a vehicle for injections and of protecting the cell preparation during transport.
As is known, hyaluronic acid plays a vital role in many biological processes such as tissue hydration, proteoglycan organization, cell differentiation, proliferation and angiogenesis (J. Aigner et al. L. Biomed. Mater. Res. 1998, 42, 172-181). Hyaluronic acid derivatives maintain all the properties of said glycosaminoglycan, with the advantage of being able to be processed in various forms and having solubility and degradation times which vary according to the type and percentage of derivation (EP 0216453 B1). Moreover, the hyaluronic acid derivatives offer new properties due to the insertion of specific molecules in the structure of the hyaluronic acid. For example, the sulfated derivatives of hyaluronic acid have anticoagulant properties and are resistant to hyaluronidase (WO 95/25751). It has been demonstrated that said compositions do not trigger immune responses by the organism and the chondrocytes they contain maintain their phenotype. Hyaluronic acid derivatives are not cytotoxic and allow the synthesis of components of the extracellular matrix that are necessary for the development of the cartilage tissue. Moreover, said derivatives do not represent a simple vehicle for the cells but are able to stimulate their poliferation and, as they degrade, allow the development of the cells into three-dimensional structures. Besides stimulating the growth of implanted cells, the hyaluronic acid derivatives are able to create an extracellular environment similar to that of mammal foetuses which stimulates the regeneration of tissues. Moreover, as the hyaluronic acid derivatives degrade, they release oligomers, stimulating the recruitment of progenitor cells of chondrocytes and favouring their development towards the chondrocyte cell line. Such hyaluronic acid derivatives have been proposed for use in treatment of arthropathies (WO 97/49412).
It is known that hyaluronic acid derivatives can be used as three-dimensional, solid scaffolds in the form of non-woven fabrics, sponges, granules, microspheres, tubes and gauzes to grow stem cells in vitro (WO 97/18842), in the form of non-woven fabrics associated with a perforated membrane for the growth in vitro of fibroblasts and keratinocytes (WO 96/33750) and in the form of non-woven fabrics for the growth of chondrocytes (J. Aigneretal., L. Biomed. Mater. Res., 1998, 42, 172-181). However, to date, nobody has made an injectable gel containing hyaluronic acid derivatives and mammalian cells, such as chondrocyte cells, that allows the surgeon to use only mildly invasive surgical techniques, such as endoscopic surgery, enabling the cells to be incorporated in a composition to survive transport and completely fill irregularly-shaped lesion sites.
Unlike the method of seeding of cells on solid supports, in the present invention the cells are evenly dispersed in all three dimensions throughout the composition in the form of a gel made according to the present invention. Said compositions allow the regenerated tissue to integrate perfectly with the cartilage tissue surrounding the defect. The compositions according to the present invention can be used to advantage for the treatment of both superficial and deep cartilage defects. Superficial defects are those affecting the cartilage tissue alone, while deep defects are those which also involve the subchondral bone tissue and the layer of calcified cartilage between the subchondral bone tissue and the cartilage.
The present invention concerns injectable, biocompatible and biodegradable compositions containing at least one hyaluronic acid benzyl ester derivative and/or auto-cross-linked derivative, at least one pharmacologically and/or biologically active substance, such as a growth factor, and/or at least one mammalian cell, particularly chondrogenic cells.
1. The Hyaluronic Acid Component
The present invention, therefore, describes injectable biocompatible compositions based on a benzyl ester of hyaluronic acid or on an auto-cross-linked derivative of hyaluronic acid, used singly or in mixtures with one another, characterized by high biocompatibility. The materials are also completely biodegradable and do not need to be removed from the application site, thus avoiding a second surgical operation. When prepared in the form of gels, the cross-linked derivatives present materials with significantly greater viscosity than the unmodified polymer and with variable degradation times.
The term xe2x80x9chyaluronic acidxe2x80x9d is used in literature to designate an acidic polysaccharide with various molecular weights constituted by resides of D-glucuronic acid and N-acetyl-D-glucosamine, which naturally occur in cellular surfaces, in the basic extracellular substances of the connective tissues of vertebrates, in the synovial fluid of joints, in the vitreous humor of the eye, in the tissue of the human umbilical cord and in cocks"" comb.
Hyaluronic acid plays an important role in the biological organism, firstly as a mechanical support of the cells of many tissues, such as the skin, the tendons, the muscles and cartilage and it is therefore the main component of the extracellular matrix. But hyaluronic acid also performs other functions in the biological processes, such as the hydration of tissues, lubrication, cellular migration, cell function and differentiation. (See for example, A. Balazs et al., Cosmetics and Toiletries, No. 5/84, pages 8-17). Hyaluronic acid may be extracted from the above-mentioned natural tissues, such as cocks"" combs, or also from certain bacteria.
Today, hyaluronic acid may also be prepared by microbiological methods. The molecular weight of whole hyaluronic acid obtained by extraction is in the region of 8-13 million Daltons. However, the molecular chain of the polysaccharide can be degraded quite easily under the influence of various physical and chemical factors, such as mechanical influences or under the influence of radiation, hydrolyzing, oxidizing or enzymatic agents. For this reason, often in the ordinary purification procedures of original extracts, degraded fractions with a lower molecular weight are obtained. (See Balazs et al., cited above). Hyaluronic acid, its molecular fractions and the respective salts have been used as medicaments and their use is also proposed in cosmetics (see for example, the above-mentioned article by Balazs et al., and the French Patent No. 2478468).
Although the term xe2x80x9chyaluronic acidxe2x80x9d is commonly used in an improper sense, meaning, as can be seen from above, a whole series of polysaccharides with alternations of residues of D-glucuronic acid and N-acetyl-D-glucosamine with varying molecular weights or even degraded fractions of the same, and although the plural form xe2x80x9chyaluronic acidsxe2x80x9d may seem more appropriate, the discussion herein shall continue to use the singular form to refer to hyaluronic acid in its various forms including its molecular fractions.
The present invention describes injectable compositions containing hyaluronic acid derivatives which work as suitable carriers for biological/pharmacological cells or molecules. Hyaluronic acid derivatives are certainly more suitable than other biomaterials/scaffolds known in the prior art. In comparison with biological-derived system, such as, for instance, cadaveric acellular material, hyaluronic acid has the advantage to be readily available in unlimited supply and not highly immunogenic, such as allogeneic donor tissues. In addition, hyaluronic acid is not at risk for cross-contamination for infective diseases, especially virus derived (HIV, Hepatitis, etc.). In comparison with more purified biological-derived molecules, such as, for instance collagen, proteoglycans and fibrin, or biocompatible synthetic polymers, such as, for instance, PLLA/PGA, PTFE, hyaluronic acid has different favourable characteristics. First of all, hyaluronic acid is a polysaccharide which shows less immunogenic reactions than common proteic- of proteic-based compounds. Secondly, hyaluronic acid is commonly found in all mammals species with no modification of the molecular structure, thus, is very well known and tolerated by the human body. Third, hyaluronic acid has many biological effects, in developing as well as adult humans, which make the molecule to be fundamental in each reparative/regenerative process. Finally, another favourable point is that hyaluronic acid is present in almost all tissues/organs of the human body, being a major component of the extracellular matrix. This fact, along with the simple composition of the polymer, make hyaluronic acid different from many proteic extracellular matrix molecules, such as, for instance collagen, that are, very often, tissue/organ specific. This last point is very important in designing a general and biocompatible delivery vehicle to be used for different compartment of the human body.
2. The Benzyl Ester Derivatives
The first preferred material of the invention is based on the benzyl ester of hyaluronic acid, particularly the 50-75% esters wherein 50% to 75% of the hyaluronic acid carboxyl groups are esterified with a benzyl residue. Those benzyl esters wherein 50-75% of the hyaluronic acid carboxyl groups are esterified with a benzyl group are referred to as xe2x80x9cpartial estersxe2x80x9d, because only a portion of the carboxyl groups are esterified and the remaining carboxyl groups are either free or salified with an alkaline or alkaline earth metal, such as sodium, calcium or potassium.
Most preferred for the compositions of the invention are the benzyl esters wherein 50% of the hyaluronic acid carboxy groups are esterified. The benzyl esters of hyaluronic acid according to the invention may be prepared by methods known per se for the esterification of carboxylic acids, for example by treatment of free hyaluronic acid with the alcohol (benzyl alcohol) in the presence of catalyzing substances, such as strong inorganic acids or ionic exchangers of the acid type, or with an etherifying agent capable of introducing the desired alcoholic residue in the presence of inorganic or organic bases.
The benzyl hyaluronic esters may, however, be preferably prepared to advantage according to a particular method described in EP 0 216 453. This method consists of treating a quaternary ammonium salt of hyaluronic acid with an etherifying agent, preferably in an aprotic organic solvent.
For the preparation of the benzyl esters it is possible to use hyaluronic acids of any origin, such as for example, the acids extracted from the above mentioned natural starting materials, for example, from cocks"" combs. The preparation of such acids is described in literature; preferably, purified hyaluronic acids are used. According to the invention, especially used are hyaluronic acids comprising molecular fractions of the integral acids obtained directly by extraction of the organic materials with molecular weights varying within a wide range, for example, from about 90%-80% (M=11.7-10.4 million Daltons) to 0.2% (M=30,000 Daltons) of the molecular weight of the integral acid having a molecular weight of 13 million Daltons, preferably between 5% and 0.2%. Such fractions may be obtained with various procedures described in literature, such as by hydrolyzing, oxidizing, enzymatic or physical procedures, such as mechanical or radiational procedures. Primordial extracts are therefore often formed during these same purification procedures (for example, see the article by Balazs et al., quoted above in xe2x80x9cCosmetics and Toiletriesxe2x80x9d). The separation and purification of the molecular fractions obtained are brought about by known techniques, for example by molecular filtration.
One fraction of purified hyaluronic acid suitable for use according to the invention is for example that known as xe2x80x9cnon-inflammatory-NIF-NaHAxe2x80x9d sodium hyaluronate described by Balazs in the booklet xe2x80x9cHealonxe2x80x9dxe2x80x94A guide to its use in Ophthalmic Surgery, D. Miller and R. Stegmann, eds. John Wiley and Sons, N.Y., 81983: p 5.
Particularly important as starting materials for the benzyl ester are two purified fractions obtainable from hyaluronic acid, for example the ones extracted from cocks"" combs, known as xe2x80x9cHyalastinexe2x80x9d and xe2x80x9cHyalectinxe2x80x9d. The fraction Hyalastine has an average molecular weight of about 50,000 to 100,000 Daltons while the fraction Hyalectin has an average molecular weight of between about 500,000 and 730,000 Daltons. A combined fraction of these two fractions has also been isolated and characterized as having an average molecular weight of about 250,000 to about 350,000 Daltons. This combined fraction may be obtained with a yield of 80% of total hyaluronic acid available in the particular starting material, while the fraction Hyalectin may be obtained with a yield of 30% and the fraction Hyalastine with a yield of 50% of the starting hyaluronic acid. The preparation of these fractions is described in EP 0 138 572.
The following Examples describe the preparation of the benzyl esters of hyaluronic acid.